Abstract A75: Synergy between conventional chemotherapy and Cdc7 inhibition as a novel approach for cancer therapy.

Abstract

Abstract Cell division cycle 7-related protein kinase (Cdc7) is a heterodimer of a kinase and its activator (Dbf4) and is essential for the activation of the minichromosome maintenance complex (MCM2–7), the helicase that unwinds the strands of DNA during replication. This kinase as well as its substrate have been shown to be overexpressed in many different tumors including the majority of both solid tumors and hematologic malignancies. For this reason, it represents a potential novel target for a cytotoxic chemotherapeutic agent. Previous work in our laboratory used a high throughput screen to identify compounds that inhibit Cdc7 kinase activity. MSK-747 and 3 of its naturally occurring derivatives, including MSK-777, were identified as lead compounds based on their potency, and MSK-777 was chosen as the lead compound for further preclinical and clinical development. While we are completing the FDA required studies needed to move MSK-777 to a Phase I clinical trial, we have begun work to determine an optimal Phase II regimen. Time course experiments have been performed to determine if synergy exists between known anti-leukemic chemotherapies such as etoposide, cytarabine, daunorubicin, or hydroxyurea and MSK-777. Cells were collected at time points for viable cell count, fluorescence-activated cell sorting (FACS) for DNA content, Western blotting, and caspase-3 activation assay. Multiple drug titration experiments were performed using daunorubicin, etoposide, cytarabine, and hydroxyurea in order to determine the optimal dosing of the drugs. Experiments using daunorubicin and cytarabine were performed where the standard chemotherapies were given prior to, concurrent with, and following MSK-777. Based on FACS and cell viability, it did not appear that either daunorubicin or cytarabine were synergistic with MSK-777. Etoposide caused the cells to arrest in S phase, and this arrest persisted for at least 48 hours. Based on the mechanism of action of MSK-777, etoposide was administered as a 12 hour pulse and then the cells were released into MSK-777. On FACS, at 24 hours the cells that had been treated with this combination continued to demonstrate the presence of a G1 population that was not seen with either agent alone, but by 48 hours this population no longer existed as the cells began to undergo apoptotic cell death. By 60 hours, cell death was evident by both FACS analysis and cell viability. Significant caspase-3 activation was seen at 36 hours in the cells that had received the combination therapy as compared with the controls. Given that hydroxyurea interferes with the G1/S phase progression, it was hypothesized to potentially be a more synergistic combination. Hydroxyurea was also administered as a 12 hour pulse prior to exposing the cells to MSK-777. On FACS, G1/S phase arrest is evident at 24 hours. This arrest persists with significant cell death at 60 hours being evident by cell viability, FACS analysis, and caspase-3 activation assay. This regimen demonstrated the highest degree of potential synergy. We will present data on the mechanism of this synergy and its use in primary patient samples of leukemia. Based on these results, the combination of hydroxyurea and MSK-777 is a promising regimen to be explored in further pre-clinical experiments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A75.